The Function And Conformational Changes Of TM 3-4 Loop And HP2 Of EAAT2 During The Transport Cycle

Glutamate is the most important excitatory transmitter in the mammalian brain.It is continuously released from the cell,and then is removed by reuptake outside the cell to maintain a very low extracellular concentration to avoid the excitotoxicity of glutamate to the cell.The realization of this basic neural function depends on the excitatory amino acid transporter(excitatory amino acid transporters,EAATs)expressed on the plasma membrane of glial cells and neurons.Although both glial cells and neurons express EAATs,it is currently believed that EAATs expressed in glial cells has a stronger ability to transport glutamate than neurons,especially the astrocyte glutamate transporter subtype EAAT2 mediates 90%of the absorption of glutamate in the brain.As an important subtype of EAATs,EAAT2 is highly expressed in neurons in the cerebellum,hippocampus and even the entire brain.Because EAAT2 is highly expressed at the axon ends of hippocampal neurons,it is considered to play an important role in maintaining synaptic glutamate homeostasis and regulating mitochondrial function.In recent years,some studies have reported that the abnormal function or expression of EAAT2 is involved in the pathogenesis of various mental and neurological diseases,such as Parkinson's disease(Parkinson's disease,PD),Alzheimer's disease(Alzheimer's disease,AD),Huntington's disease(Huntington's disease,HD),Amyotrophic Lateral Sclerosis(Amyotrophic lateral sclerosis,ALS),Epilepsy and Traumatic Brain Injury(Traumatic brain injury,TBI),etc.In addition,the transport of glutamate by EAATs is electrogenic,involving the co-transportation of 3 Na⁺and 1 H⁺and the reverse transport of 1 K⁺.The latest advances in the transport mechanism and pharmacology of EAATs are based on the analysis of the crystal structure of its prokaryotic homologue Glt_Ph and the eukaryotic subtype EAATlcryst which consists of three identical subunit monomers,each of which contains eight transmembrane domains(Transmembrane domain,TM)1-8 and two A highly conservative reentrant spiral hairpin structure(hairpin loop 1,HP1 and hairpin loop 2,HP2).TM1-TM6 form an amino terminal cylinder surrounding the core of the transporter and provide structural support for the conformational changes of the transporter,while the more conserved TM7 and TM8 and HP1 and HP2 form the core of the transporter and are responsible for binding to substrates and ions.HP2 is the extracellular gating involved in the substrate response,while HP1 forms the intracellular gating.Through the study of the prokaryotic crystal structure,the TM3-4 loop undergoes a substrate-sensitive conformational change during the transportation process,which is an important part of the transportation mechanism.Moreover,the information on the structure and function of the TM3-4 loop revealed by the crystal structure of EAAT1_cryst is very limited.Based on the results of our previous studies,we found that EAAT2 TM2 plays an important role in the process of glutamate transport.Based on the special position of residues in TM2 and the important functions negative during the transport process,we believe that this transmembrane domain may be involved in the composition of the EAAT2 anion permeation pathway.However,the mechanism of anion penetration of EAATs is still not completely clear.This subject mainly uses gene-directed mutations and electrophysiological methods to reveal the relationship between the TM3-4 loop and the substrate transport channel and the negative function in substrate transport,and clarify the important role of TM2 in the anion permeation mechanism of EAATs,and provide a theoretical basis for thoroughly elucidating the transport mechanism of EAATs.1.Relative movement of TM3-4 loop and HP2 during EAAT2 substrate transportIn order to explore the spatial position and function of the TM3-4 loop in the transport cycle,we introduced several paired cysteine mutants between the TM3-4 loop and HP2 in the cysteine-free EAAT2(cysteine-less EAAT2,CL-EAAT2).We observed that Cu(?)(1,10-phenanthroline)3(CuPh)has a significant transport inhibitory effect on the A167C/G437C mutant,while dithiothreitol(dithiothreitol,DTT)reverses the oxidative cross-linking effect on A167C The effect of/G437C on the transport activity confirms that the effect of CuPh on the mutant is due to the formation of disulfide bonds in the transporter molecule.We observed that the V_max of A167C/G437C decreased,while the K_m was significantly increased.The results of kinetic parameters further showed that the formation of disulfide bonds impaired the transport activity of A167C/G437C,and the affinity of A167C/G437C to the substrate was significantly down-regulated during transportation,the A167 or G437 site may be related to the binding of the substrate.D,L-threo-?-benzyloxy-aspartic acid(D,L-threo-benzyloxy aspartate,D,L-TBOA)attenuates the CuPh inhibitory effect of the A167C/G437C mutant,while L-glutamate or KCl enhances the CuPh inhibitory effect of the A167C/G437C mutant,indicating The A167C and G437C cysteines are far apart in the outward conformation and closer in the inward conformation.The evidence we provide shows that the TM3-4 ring and HP2 change their spatial proximity during the transportation cycle.In addition,we found that when the transporter adopts an outward conformation during the transport cycle,the water permeability of G437C residues is reduced,which indicates that G437C has conformational sensitivity and may play a role in the transport cycle of EAAT2.In addition,we demonstrated the water permeability of MTSET to A167C and G437C,which clearly shows that G437 is located outside the cell.All in all,our work shows that the TM3-4 loop and HP2 may be very close during the transport cycle,the TM3-4 loop may play an important role in substrate translocation,and the TM3-4 loop and HP2 may interact to regulate the transporter.Conformational changes in binding to the substrate.2.The Effect of TM2 on the Function of Anion Channels in the Process of EAAT2 Substrate TransportIn order to study the effect of key amino acid residues in TM2 on the function of EAAT2 anion channel during transport,we transiently expressed EAAT2 and its mutants in HeLa cells,and measured EAAT2 related currents by whole-cell patch clamp experiments.We found that after the addition of 0.5 mmol/L glutamate,P95A caused a significant change in the time and voltage dependence of the EAAT2 current,while the currents of the R87K,R87S and K90R mutants did not show this dependence.Compared with WT EAAT2,after replacing part of Cl^-solution with SCN^-solution,the amplitude of current change between the presence and absence of L-glutamic acid in P95A EAAT2 was significantly reduced and compared with WT EAAT2,the anion current of P95A was significantly reduced,which indicates that P95A reduces the anion current associated with EAAT2.Combined with the results of our previous research,it is shown that the structural and property changes caused by the alanine mutation at the TM2 P95 site of EAAT2 are related to the changes in the properties of the anion channel associated with EAAT2.